Recombinant DNA technologies and molecular biological methods of cloning nucleic acids are known in the art. Such methods include manipulating nucleic acids using restriction endonucleases, ligases, nucleotide/nucleoside kinases and phosphatases, polymerases, and other molecular biology tools to generate desired recombinant nucleic acids. Several laboratory manuals have been written as reference books for molecular biology researchers. Examples of these reference manuals include, Sambrook, J., E. F. Fritsch and T. Maniatis, 1989. Molecular Cloning: A Laboratory Manual, 2nd. ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press; and Joseph Sambrook and David Russell, 2001. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press.
A problem in the art is the lack of methodology to engineer chimeric protein expression products where modular elements can be easily inserted at desired positions within a chimera. In general, while hybrid or chimeric proteins have been made successfully, the goal of synthesis has been towards final endpoint products, without building-in a predetermined mechanism to add more modules. An aspect of the invention addresses this problem by contemplating the future need for chimeras and variations thereof and making it possible to build them without starting from scratch for each one. One embodiment of the invention generally relates to methods of making fusion protein expression products. Another embodiment of the invention relates to methods of making fusion protein expression products with pre-engineered modules. Another aspect of the invention relates to building block molecules or modules, where modules are pre-designed to be capable of insertion into a chimeric protein expression cassette. The instant invention also contemplates libraries of engineered modules that can be utilized in the disclosed methods. In one embodiment, modules are made with predetermined restriction sites.